CHIRAL CALIXARENES AS POTENTIAL ENANTIOSPECIFIC STATIONARY
PHASES IN CAPILLARY GAS CHROMATOGRAPHY
by
RONNIE HEFLEY
Submitted in Partial Fulfillment ofthe Requirements
for the Degree of
Master ofScience
in the
Chemistry
Program
YOUNGSTOWN STATE UNIVERSITY
June, 2000
Chiral Calixarenes as Potential Enantiospecific Stationary Phases in
Capillary Gas Chromatography
RONNIE HEFLEY
I hereby release this to the public. I understand this thesis will be housed at the
Circulation Desk ofthe University Library and will be available for public access. I also
authorize the University and other individuals to make copies ofthis thesis as needed for
scholarly research.
Signature:
Approvals:
~~./~.~~~e, Ph.D., Thesis Advisor Date
~~'--~' c-~'o-mm-itt-ee-M-em-b-er--------.r:::.k+~~af-'-f.v""'""? ...::....-
Steven M. Schildcrout, Ph.D., Committee Member
Peter J. Kasvinsky, Ph.D., Dean ofGrad
Date
111
Abstract
The ability ofa chiral calix(4)arene to participate in host-guest interactions could
ultimately lead to new types ofstationary phases for performing enantiomeric
separations in capillary gas chromatography. For this work, chirality was introduced into
p-tertbutylcalix(4)arene via addition ofL-phenylalanine to the hydroxyl groups located at
the bottom ofthe cup-like structure. The amino-acid derived p-tertbutylcalix(4)arene
was absorbed into the polymeric coating ofa non-polar siloxane AT-1 stationary phase
using evaporation under reduced pressure. Solute retention times were plotted versus the
number ofalkyl chain carbon atoms in a homologous series ofalkyl benzenes.
Deviations in the linearity ofthis plot suggested some type ofcontribution to solute
retention was resulting, possibly from inclusion ofthe component(s) into the calixarene
cavity. Indications ofpossible host-guest interactions between the amino acid-derived
stationary phase with various analytes were apparent from the band broadening due to the
slow kinetics, which are typically observed with host-guest interaction. The ability of
this newly derived stationary phase to selectively interact with analytes based on their
relative polarity was also determined.
IV
Acknowledgements
I would like to thank Dr. James Mike and Dr. Daryl Mincey for providing me
with this opportunity. Without the direction and encouragement provided by Dr. Mike, I
would never have completed this program. I would also like to express my sincere love
and appreciation to Carrie and Dr. Larry Curtin, without your friendship and food I would
not have survived my first year here. Also, thank you to Dr. Shildcrout and Dr. Curtin
for their contributions to this manuscript. Finally, I would like to express my deepest
appreciation to Dr. Bruce Levison, who sacrificed endless hours to help make_this project
a success. Thank you for all ofthe knowledge and patience you provided me.
In every way possible, Bruce went above and beyond the call ofduty to help me. I hope
some day I can return that favor by helping you.
Table ofContents
TITLE PAGE
SIGNATURE PAGE
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER
I. Introduction
A. Gas Chromatography
B. Detection ofSolutes in GC
C. Peak Broadening and Component Retention
D. Host-Guest Complexation
E. Calixarenes
II. Statement ofthe Problem
III. Literature Review
IV. Materials and Methods
A. Materials
B. Methods
1. Synthesis ofthe Chiral Calixarene
2. Absorption into the Stationary Phase
111
IV
V
Vll
V111
IX
1
1
1
2
8
9
12
14
18
18
19
19
22
v
Table ofContents (cont.)
3. Evacuation ofthe Solvent 23
4. Conditioning the Column 24
5. Descriptions ofPrepared Stationary Phases 24
6. GC Parameters 24
7. GC Experiments 25
V. Results and Discussion 27
A. Characterization ofthe Phenylalanine-Derived Calix(4)arene 27
B. Absorbed Stationary Phases 30
C. Homologous Series ofAlkyl Benzenes 33
D. Homologous Series ofAlcohols 34
E. Phenolic Mixture 38
F. Isomeric Mixtures 38
G. Mixture ofa- and p-Napthol 44
VI. Conclusions 47
VII. Future Work 48
REFERENCES 50
VI
List ofTables
TABLE
1. Typical Chromatographic Conditions
2. Component Retention Data
3. Asymmetry Factors
VB
PAGE
25
37
41
List ofFigures
FIGURE
1. Structure of4-tert-butylcalix(4)arene
2. Diagram ofabsorption process
3. Synthesis ofamino acid-derived calix(4)arene
4. Diagram ofcolumn filling apparatus
5. IH NMR ofunhydrolyzed amino acid-derived calix(4)arene
6. Mass spectrum ofunhydrolyzed product
7. IH NMR ofhydrolyzed amino acid-derived calix(4)arene
8. Mass spectrum ofhydrolyzed product
9. Effects ofhost-guest complexation upon correlation or RHS at 90?C
10. Effects ofhost-guest complexation upon correlation ofAHS at 90?C
11. Retention ofphenol (1), resorcinol (2), & tert-butylphenol (3) at 80?C
12. Retention oftert-butanol, sec-butanol, and n-butanol (respectively) at 35?C
13. Retention of(-)-menthol at 90?C
14. Retention of2-methyl-2-butanol through 2-pentanol (respectively) at 35?C
15. Retention ofa-napthol (1) and p-napthol (2) at 90?C
Vlll
PAGE
10
11
20
23
28
29
31
32
35
36
39
40
42
43
46
AHS
a
A
APCI
amu
k'
!1Z
CSP
to
DIPEA
DMF
ECD
FID
FPD
F
GC
HATU
HETP
J.D.
I
IX
List ofAbbreviations
Alcohol Homologous Series
Alpha
Asymmetry factor
Atmospheric Pressure Chemical Ionization
Atomic mass unit
Beta
Capacity factor
Change in tr between solutes
Chiral Stationary Phase
Dead time (min)
Diisopropylethylamine
N,N-Dimethylformamide
Electron Capture Detector
Flame Ionization Detector
Flame Photometric Detector
Flow rate
Gas Chromatography
0- (7-Azabenzotriazol-I-yl)-N, N, N', N' 
hexafluorophosphate
Height equivalent to a theoretical plate
Internal diameter
Length ofcolumn (m)
x
List ofAbbreviations (cont.)
u Linear velocity
m/z Mass-to-charge ratio
ilL Microliter
Ilm Micrometer
Ils Microsecond
mg Milligram
mL Milliliter
mm Minute
mol Mole
NMR Nuclear Magnetic Resonance
W Peak width
pSI Pounds per square inch
rc Column radius (cm)
RHS Reference Homologous Series
Rs Resolution
tr Solute retention time (min)
THF Tetrahydrofuran
TCD Thermal Conductivity Detector
TLC Thin Layer Chromatography
TFA Trifluoroacetic acid
1
CHAPTER I
Introduction
A. Gas Chromatography
Chromatography is the general name given to the methods by which two or more
compounds in a mixture physically separate by distributing themselves between a
stationary and a mobile phase. The stationary phase can be a solid or a liquid supported
on a solid and the mobile phase can be either a gas or a liquid, which flows continuously
through the stationary phase. Gas chromatography (GC) is a mode ofpartioning
chromatography where the mobile phase is a gas. GC is the most widely used method for
separation ofmixtures ofgases or for volatile liquids and solids because the separation
times are on the order ofa matter ofminutes, even for very complex mixtures.
B. Detection ofSolutes in GC
After GC separates the components ofa mixture, they must be detected as they
exit the column. The requirements ofa GC detector depend on the separation
application, which is dependent upon the selectivity ofthe analysis. For example, one
analysis might require a detector that is selective for chlorine-containing compounds.
These methods can be either destructive, where the analyte cannot be recovered for
further spectroscopic analysis, or it can be nondestructive, where the analyte can be
recovered for further spectroscopic analysis.
Thermal-conductivity (TCD) and flame-ionization (FID) detectors are the two
most common detectors in GC. A TCD consists ofan electrically heated wire or
thermistor, where the temperature ofthe sensing element depends on the thermal
2
conductivity ofthe gas flowing around it. When organic molecules displace the carrier
gas, the changes in thermal conductivity cause a temperature rise in the element, which is
sensed as a change in resistance. A TCD is not as sensitive as other detectors, but is non 
specific and nondestructive. A FID consists ofa hydrogen/air flame and a collector plate,
where ions are collected and produce an electrical signal. When the eluant from the GC
passes through the flame, the organic molecules are broken down, producing ions. The
ions are collected on an electrode and produce an electric signal. The FID is extremely
sensitive, but it destroys the sample. Another widely used type ofdetector is an electron 
capture detector (ECD). The ECD uses a radioactive p- emitter to ionize some ofthe
carrier gas and produce a current between a pair ofelectrodes. When organic molecules
that contain electronegative functional groups pass through the detector, those functional
groups capture some ofthe electrons and reduce the current measured between the
electrodes. The ECD is extremely sensitive, but is limited to the analysis ofhalogenated
compounds. Mass spectrometers (MS) are also widely used as a method ofdetection for
GC. MS use the difference in mass-to-charge ratio (m/z) ofionized atoms or molecules
to separate them from each other. MS is therefore useful for quantification ofatoms or
molecules and also for determining chemical and structural information about the
molecules, such as their fragmentation patterns.
C. Peak Broadening and Component Retention
Peak broadening and retention are behavior characteristics unique to each
component ofa mixture in gas chromatography. The time and manner in which a
particular compound elutes is a "fingerprint" characteristic that can be used to help
3
identify it in a mixture. Resolution and separation ofthose components are generally
controlled by both thermodynamic and kinetic parameters. The thermodynamic and
kinetic factors that determine retention times are fundamental properties ofa solute
relative to the stationary phase. Optimization ofthese effects results in enhanced
chromatographic separations.
The GC oven controls the temperature ofthe column. Chromatographic
separations can be carried out under isothermal conditions, or it is possible to perform
temperature gradient separations, where the oven is programmed to change the column
temperature at some predictable and reproducible rate (OC/min). A temperature gradient
can be especially useful in separating mixtures ofsubstances with widely varying heats of
-vaporization. The linear velocity ( u ) and the flow rate (F) are two common factors that
depend upon the head pressure ofthe carrier gas. The linear velocity ofthe carrier gas
is described by:
Iu
The flow rate ofmobile phase through the column is described by:
F
where: I = column length (em) and rc = radius ofthe column (em). The dead time (to) is
the amount oftime that it takes for an unretained component to elute from the column
and is thus dependent on head pressure ofthe mobile phase (carrier gas). These factors
serve as a reference point for assisting in the determination ofthe identity ofthe
components in a mixture. With these established parameters in place, the identity ofa
4
compound can be determined by comparison ofthe experimental retention time versus
the literature value for the retention time ofthat particular component, or versus a
standard mixture ofvarious components.
Peak resolution is a function ofboth the solute distribution coefficient, Ko,
between the mobile and stationary phases and the ratio ofthe volumes, <1>, ofmobile and
stationary phases in the column. It is possible to experimentally determine the
distribution coefficient, but the phase volume ratio is not easily obtainable because ofthe
nature ofthe stationary phase. Thus, because retention time is a combination ofthese
two factors, a ratio, called the capacity factor (k'), is used to describe chromatographic
retention. The capacity factor is defined by:
k' !r ~to
to
where tr is the component retention time and to is the dead time ofthe column. Small
values for the capacity factor indicate little interaction between component and stationary
phase resulting in the elution ofsolutes close to the umetained peak. Larger values for
the capacity factor indicate more interaction between component and stationary phase
resulting in longer retention times.
A common thermodynamic description used to characterize the partitioning
process ofsolutes during chromatography can be expressed in terms ofthe system free
energy, where:
!1GO =MIO -T!1S0 =-RTlnK
and:
k'K= 
cD
or:
therefore:
I1GO k'InK
= - = In- = Ink' -In<DRT <D
I1GOIn k' =- + In <I>
RT
5
where K is the equilibrium constant, k' is the solute capacity factor, ~Go is the free
energy ofpartitioning, ~H and ~s are the enthalpy and entropy ofpartitioning ofthe
solute from the mobile to the stationary phase, R is the universal gas constant, T is the
temperature (in Kelvin), and <D is the phase volume ratio (i.e., the volume ofthe
stationary and mobile phases relative to one another).
If:
where i1G is the free energy contribution for each ofn carbon atoms in a molecule or
chain and b is some constant independent ofn, then a plot ofIn k' versus n at a constant T
will have a slope of:
dInk'
dn
I1G---
RT > 0 if i1G < 0 for adsorption.
Therefore, a constant contribution for each ofn carbon atoms in a molecule or chain of
homologous series would result in a straight line plot.
Gas chromatography is a dynamic process, which includes mass-transport that can
be described kinetically. These effects are described using the van Deemter equation.
Peak broadening during a chromatographic separation can be attributed to a number of
random molecular processes that can be grouped as either non-column or column effects.
6
Non-column peak broadening is due to the dispersion ofcomponent molecules in
the carrier gas in the injector and connections before the column and between the column
and detector. Dispersion is most significant in open tubular GC columns, but can be
avoided by good installation and sealed connections between injectors, detectors, tubing,
and fittings. Minimization ofthe dead volume will lead to optimized resolution of
components.
Column peak broadening for chromatography was shown by van Deemter to be
the summation ofthe result ofthree molecular diffusion processes. The van Deemter
equation expresses this in terms ofHETP, the height equivalent to a theoretical plate:
HETP = A + B + Cu
u
where A, B, and C are constants related to the three major factors affecting the HETP. A
represents eddy diffusion and is due to the variety ofpathways available between the
particles in the column and is independent ofthe carrier gas velocity. Eddy diffusion is a
characteristic ofthe column packing and can be decreased with smaller and more uniform
particles and tighter packing. This results in increased efficiency ofthe column. Good
separation and minimum peak broadening will be achieved using small particles with a
narrow size range that are packed uniformly into the column, however, an extremely
small support is difficult to pack uniformly. B represents the longitudinal or molecular
diffusion ofthe sample components in the carrier gas due to the concentration gradients
inside the column. High flow rates reduce molecular diffusion, which can be changed
only by varying the pressure, flow rate, and type ofcarrier gas. C represents the rate of
mass transfer and is due to the amount oftime taken for solute equilibrium to be
7
established between the mobile and stationary phases. Increasing the temperature, which
effectively increases the solubility ofthe vapor components ofthe sample decreases the
rate ofmass transfer. Also, by decreasing the flow rate more time is allowed for the
solute equilibrium to be established, which results in a decreased rate ofmass transfer.
All conditions must be adjusted to obtain a balance between molecular diffusion and
mass transfer. The three molecular diffusion processes are kept as small as possible to
provide the minimum HETP or an increase in efficiency for the sample solute.
With respect to polarity effects, this kinetic dependence results in the response of
the FID being proportional to the number ofcarbon atoms. This response diminishes
with increasing substitution by halogens, amines, or hydroxyl groups. Other kinetic
interactions that could affect the response ofthe detector include either some type of
chemical reaction occurring between the solute and the stationary phase or an interaction,
based on something outside polarity effects occurring between the solute and the
stationary phase. A chemical reaction occurring in the column is not desirable because it
changes the composition ofthe eluting component and the chemical behavior ofthe
stationary phase. Some type ofhost-guest interaction based on an effect outside ofthose
due to polarity could result in a more specific separation without affecting the
composition ofthe eluting component or the chemical behavior ofthe stationary phase.
Peak resolution is defined simply as the degree ofseparation between two
adjacent peaks. The resolution Rs ofa column provides a quantitative measure ofits
ability to separate two components and is defined as:
2 [(tr ) B - (tr ) A]
WA -WB
8
where tr is the retention time for components A and Band W is the width at the base of
the peak for components A and B. Rs < 1 means the peaks are not resolved; Rs = I means
the peaks are resolved well enough for quantitative analysis, but are not baseline
separated; Rs > 1 means the peaks are baseline resolved.
Peak resolution is strongly affected by peak symmetry. In chromatography, it is
assumed that peaks emerge with either a Gaussian or Poisson type concentration profile.
The asymmetry factor (A) mathematically describes the symmetry ofthe peak and is
defined by:
A b
a
where b is the distance after the peak center and a is the distance before the peak center to
the detector response measured at 10% ofthe total peak height. A value ofone would
indicate a mathematically symmetric peak; values less than one result from fronting
peaks; values larger than one result from tailing peaks. Any host-guest interactions that
are additional to bulk absorption result in an increased retention time. The kinetics ofthis
interaction would be slower than the kinetics ofthe absorption, which is called mixed 
mode retention and is characterized by tailing peaks. This would also be indicated by
increased values for both the capacity and asymmetry factors.
D. Host-Guest Complexation
A type ofseparation that requires a more specific interaction than those due
strictly to polarity is the resolution ofenantiomers. A racemic mixture is composed of
components that differ only with regard to their optical activity. As a result, polarity
9
effects alone are not enough to separate and resolve this type of
mixture. Enantiomers
only interact specifically with other enantiomers. Such separations thus require an
enantiomeric stationary phase.
In chromatography, host-guest complexation was first performed using
cyclodextrins, which have similar structure and inclusion characteristics as calixarenes
(1). Studies have indicated that this host-guest complexation results in an
enantioselective stationary phase by allowing selected components to fit themselves into
the cavityofthe cyclodextrin, much like an enzyme and substrate in biochemical
systems. This enantioselectivity was demonstrated toward carbohydrates and nitrogen
containing compounds and was based on the selectivityofthe formation of
inclusion
complexes between solute and stationary phase.
E. Calixarenes
Recently, calixarenes have drawn attention in the area ofchromatographic
separations due to the host-guest interactions that this compound has been shown to form.
Calixarenes are macrocyclic phenol-formaldehyde polycondensates that possess an
interesting basket shaped intramolecular cavity (2) (Figure1).
10
HO
p-tert-butylcalix(4)arene
Figure 1. Structure of4-tert-butylcalix(4)arene
When used as components ofstationary phases in chromatography, calixarenes
are capable offorming inclusion complexes that affect the retention ofsolutes as
compared to non-derived stationary phases. Because oftheir similarity to cyclodextrins,
which have been shown to resolve enantiomers, chiral calixarenes could potentially be
used for enantiomeric separations.
Calixarenes have been regarded as a third generation ofhost compounds, after
cyclodextrins and crown ethers, because they can form inclusion complexes with ionic
and neutral molecules. They are also easily modified to give more functionalized host
calixarenes, which may have great potential in analytical chemistry (3). Calixarenes act
as a platform that can be used to support a variety offunctional groups. The resulting
calixarene derivative has similar characteristics to the compound from which it was
made. The synthesis ofchiral calixarenes is ofgreat value for the development ofa new
class ofchiral host molecule (4). This might provide a suitable enantiomerically active
stationary phase that could be used for separations ofoptical isomers, much like the
cyclodextrins in current use in GC columns.
11
In gas chromatography, the stationary phase is most commonly a polymeric liquid
coated on the inner surface ofa capillary column. Static coating is a desirable method of
preparing a derivatized column because ofits relative simplicity.
Absortion of amino1
acid-derived calixar~ne
mixture by evaporation
under reduced pressure
Absorptionand ~~~
entanglement"":;;; (_H:_.'!(_:'If._X_'!(_H'._:'lr.-=-:'!
Figure 2. Diagram ofabsorption process
This method is done by absorption ofthe stationary phase into the nonpolar wall ofa
column containing dimethylpolysiloxane (Figure 2). It was hoped that chirality might be
introduced into the non-chiral calixarene structure through this synthetic strategy.
For the sake ofsimplicity, immobilization ofthe modified calixarene was accomplished
by absorption into the silicone polymer coating ofan Alltech AT-1 capillary column
(Figure 2).
12
CHAPTER II
Statement ofthe Problem
Gas chromatography (GC) is the method of choice for separation and analysis of
volatile or gaseous mixtures. A problem facing modem chromatographers is the
resolution of mixtures containing optical isomers (enantiomers). Chromatographic
separation of enantiomers requires the utilization ofchiral stationary phases. To prepare
these phases, chiral substrates are either attached to the inner wall of a silica capillary
column or embedded within the polymeric matrix ofan existing capillary column.
Calixarenes, a class of organic, basket shaped molecules, are promIsmg
candidates for these types of separations. They are not inherently chiral, however, and
must be modified through organic synthetic techniques to make them suitable for
potential utilization in chiral separations.
This project involves derivatizing t-butylcalix[4]arene with phenylalanine
residues through the hydroxyl groups at the bottom of the cup-like structure. The
synthetic method is based on a well characterized addition ofalanine to calix(4)arene, but
required significant modification for addition of phenylalanine. The phenylalanine 
derivatized calixarene was subsequently absorbed into a dimethylpolysiloxane (nonpolar)
stationary phase. The gas chromatographic behavior and characterization ofthe absorbed
phenylalanine-calixarene derivatives were determined by analysis of standard GC
capillary column test mixtures, two types of homologous series of compounds, isomeric
mixtures, and phenolic mixtures. For the homologous series of compounds, plotting
solute retention data (corrected retention times or capacity factors) against molecular
13
descriptors (number of carbon atoms, phenyl rings, or functional groups) helped to
provide an indication of host-guest behavior through observations of deviations from
linearity and changes in retention behavior.
14
CHAPTER III
Literature Review
Enantiomeric separations ofracemic mixtures are an ever-growing aspect ofgas
chromatography (GC). The earliest type ofenantiospecific stationary phase was based on
hydrogen bonding interactions (5). Chiral stationary phases in GC result in enantiomeric
separations with a mechanism due to solute-solvent enantioselective hydrogen bonding
interactions. Today, enantiomeric separations are still based on these interactions.
Chiral separations have many different applications; for pharmaceuticals, one-enantiomer
is usually more effective than the other. In a few instances the less effective enantiomer
even has toxic properties (6). Derivatized polysiloxanes have shown sufficient
enantioselectivity and thermal stability to separate various pharmaceuticals into
enantiomeric pairs within a reasonable time (7). Derived polysiloxanes have also been
widely used as chiral stationary phases. Polysiloxanes attached with a ~-cyclodextrin
side chain have shown good film formation, excellent isomer separation and good
enantiomeric separation ability. It was also observed that the ability ofthese derived
stationary phases to separate m- and p-chlorotoluenes decreases with a decrease in
temperature in the range of80-11 O?C (8).
The enantiomeric purity ofamino acids and the amount ofracemization in peptide
synthesis and peptide hydrolysis can easily be determined by the use ofcapillary GC on
several hydrogen-bonding chiral stationary phases (9). In 1966, Gil-Av et al. reported the
first successful separation ofenantiomers ofderivatized amino acids on a chiral amino
acid derivative (10). Later, Feibush developed chiral stationary phases containing
15
diamide moieties, which proved to be even more useful because oftheir high
enantioselectivity and reduced polarity, which lead to reduced retention times (11). A
different approach to enantiomeric separations ofamino acids derivatives by GC consists
ofthe use ofmodified cyclodextrins. Following the success ofmodified cyclodextrins as
selective stationary phases in various modes ofchromatography, calixarenes are now
considered interesting synthetic selectors in GC because oftheir high thermal stability
and their unique cavity-type supramolecular shape (2,12).
A tripeptide derivative provided excellent enantiomer separation ofa variety of
racemic mixtures, including alcohols, amines, amino acids, carboxylic acids, hydroxyl
acids, and amine acids via GC (5). Chiral recognition ofthis tripeptide derivative was
shown to be dependent upon hydrogen bonding between solutes and chiral stationary
phases (13). Other chiral stationary phases based on these same principles are being
developed and applied in all aspects ofchemistry. GC chiral separations ofseveral
derivatized trimethylsilyl ethers have shown excellent baseline separations ofthe
enantiomers, highly increased sensitivity ofdetection, no significant concentration
dependence, and decreased analysis time.
Calixarenes posses an interesting basket shaped intramolecular cavity (14). Since
they possess a cylindrical architecture similar to that ofcyclodextrins, they are expected
to form inclusion complexes (15). These compounds have been regarded as the third
generation ofhost compounds since they can complex with ionic and neutral molecules
and are easily modified to the more functionalized host calixarenes, which may have
great potential in analytical chemistry (16). When used as selective components of
stationary phases in capillary GC, calixarenes are capable offorming inclusion
16
complexes with many metal ions and organic molecules. Formation ofinclusion
complexes are determined by the overall macrocyclic structure, most importantly by the
cavity size, but also by the nature ofthe functional groups which act as the binding sites
(17). Calixarenes assume the role ofa platform supporting a variety ofligating groups,
resulting in molecules having similar characteristics to those ofthe compound from
which the calixarene derivative was made.
Until September of 1998, there had been no report on the application of
calixarenes in GC, and the reasons are due to the high melting point ofcalixarene
derivatives which leads to difficulties in coating them onto the internal was of-the
capillary column, thus resulting in poor column efficiency (18). A facile method to
resolve this problem was to attach the calixarene molecule directly onto a polymer
substrate, such as a polysiloxane. Recently, two materials were reported to have been
developed as GC stationary phases, involving a calixarene unit that was directly
dissolved into the polysiloxane or attached onto the main chain ofthe polysiloxane. The
results showed a high column efficiency and unique selectivity for aromatic isomers
(3,19). In 1977, Chirasil-Val, a chiral polysiloxane with (S)-valine-tert. -butylamide
anchored to carboxypropyl-modified dimethylpolysiloxane was reported (20). Since
then, polysiloxanes have received increasing attention as suitable liquid matrices for
anchoring and dispersing chiral moieties (6).
A series ofamino acid ester hydrochlorides bonded on a silica calix(4)arene
tetraester phase were shown to be retained in order oftheir hydrophobicity and are shown
to selectively include amines in the calixarene cavity. These amino acid-derived
calixarenes were reported to encapsulate primary amines and not secondary or tertiary
15
diamide moieties, which proved to be even more useful because oftheir high
enantioselectivity and reduced polarity, which lead to reduced retention times (11). A
different approach to enantiomeric separations ofamino acids derivatives by GC consists
ofthe use ofmodified cyclodextrins. Following the success ofmodified cyclodextrins as
selective stationary phases in various modes ofchromatography, calixarenes are now
considered interesting synthetic selectors in GC because oftheir high thermal stability
and their unique cavity-type supramolecular shape (2,12).
A tripeptide derivative provided excellent enantiomer separation ofa variety of
racemic mixtures, including alcohols, amines, amino acids, carboxylic acids, hydroxyl
acids, and amine acids via GC (5). Chiral recognition ofthis tripeptide derivative was
shown to be dependent upon hydrogen bonding between solutes and chiral stationary
phases (13). Other chiral stationary phases based on these same principles are being
developed and applied in all aspects ofchemistry. GC chiral separations ofseveral
derivatized trimethylsilyl ethers have shown excellent baseline separations ofthe
enantiomers, highly increased sensitivity ofdetection, no significant concentration
dependence, and decreased analysis time.
Calixarenes posses an interesting basket shaped intramolecular cavity (14). Since
they possess a cylindrical architecture similar to that ofcyclodextrins, they are expected
to form inclusion complexes (15). These compounds have been regarded as the third
generation ofhost compounds since they can complex with ionic and neutral molecules
and are easily modified to the more functionalized host calixarenes, which may have
great potential in analytical chemistry (16). When used as selective components of
stationary phases in capillary GC, calixarenes are capable offorming inclusion
16
complexes with many metal ions and organic molecules. Formation ofinclusion
complexes are determined by the overall macrocyclic structure, most importantly by the
cavity size, but also by the nature ofthe functional groups which act as the binding sites
(17). Calixarenes assume the role ofa platform supporting a variety ofligating groups,
resulting in molecules having similar characteristics to those ofthe compound from
which the calixarene derivative was made.
Until September of 1998, there had been no report on the application of
calixarenes in GC, and the reasons are due to the high melting point ofcalixarene
derivatives which leads to difficulties in coating them onto the internal was of-the
capillary column, thus resulting in poor column efficiency (18). A facile method to
resolve this problem was to attach the calixarene molecule directly onto a polymer
substrate, such as a polysiloxane. Recently, two materials were reported to have been
developed as GC stationary phases, involving a calixarene unit that was directly
dissolved into the polysiloxane or attached onto the main chain ofthe polysiloxane. The
results showed a high column efficiency and unique selectivity for aromatic isomers
(3,19). In 1977, Chirasil-Val, a chiral polysiloxane with (S)-valine-tert. -butylamide
anchored to carboxypropyl-modified dimethylpolysiloxane was reported (20). Since
then, polysiloxanes have received increasing attention as suitable liquid matrices for
anchoring and dispersing chiral moieties (6).
A series ofamino acid ester hydrochlorides bonded on a silica calix(4)arene
tetraester phase were shown to be retained in order oftheir hydrophobicity and are shown
to selectively include amines in the calixarene cavity. These amino acid-derived
calixarenes were reported to encapsulate primary amines and not secondary or tertiary
17
amines in the calixarene cavity, indicating a sterically controlled selectivity (21).
Transport efficiency was found to be closely related to the hydrophobicity ofthe amino
acid esters. It was postulated that the ammonium group ofthe guest is anchored in the
carrier cavity, forming an endo-calix complex (22). Since then, amino acids have been
effectively attached to the platform ofcalixarenes and homotrioxa-calix(3)arenes. It is
encouraging that the recognition, separation, and analysis ofenantiomers with this kind
ofchiral calixarene has been recently accomplished (23). These amino acid-derived
calixarenes are expected to be useful as new recognition sites in the design ofenzyme
mimics in totally synthetic systems, and it has been suggested that the synthesIs ofchiral
calixarenes would be ofgreat value for the development ofa new class ofchiral host
molecule (4).
The fundamental principles and properties ofcalixarenes and their derivatives
have shown that the simultaneous attachment and ligating functions at the upper and
lower rims leads to receptors with two different binding sites (ditopic receptors),
demonstrating the possibilities offered by calixarenes as basic platforms for GC (24).
These new molecular recognition phases based on macrocyclic calixarenes show
enormous potential for the tailoring ofchromatographic selectivity through calixarene
functionality (17).
18
CHAPTER IV
Materials and Methods
A. Materials
All reagents used in this work were ofthe highest purity available and used as
received. 4-tertButylcalix(4)arene was purchased from Fluka (Milwaukee, WI). Protein
sequencing grade TFA was purchased from Aldrich (Milwaukee, WI) and stored under
an inert atmosphere. THF was distilled over sodium metal and stored under an inert
atmosphere. NaH was a 60% dispersion in mineral oil and was purchased from Aldrich
(Milwaukee, WI). Tetramethylammomium hydroxide was a 10 wt % solution in H20
and was purchased from Aldrich (Milwaukee, WI). L-phenylalanine tert-butylester HCI
was purchased from Chem-Impex International (Wood Dale, IL) and was stored at O?C.
The solvents (HPLC grade) used throughout this work were purchased from
Fisher Scientific (Fairlawn, NJ). All gases were obtained from Praxair (Cleveland, OH).
Helium and hydrogen used as the carrier and detector gas, respectively, were ofUltra
high purity grade. Compressed air was used as an oxidizer for the Hz that was distributed
to the detector.
The AT-l fused silica capillary column (100% dimethylpolysiloxane) was
purchased as a 30m column, but was subsequently cut into 10m lengths. The column had
an J.D. ofO.25mm and a film thickness of0.25Ilm. The column ends were cut with a
ceramic scribe as straight as possible to ensure proper installation and optimum results.
The reducing unions and ferrules used were 1/16" - 1/32" and 1/32" to O.4mm,
respectively, both purchased from Valco (Deerfield, IL).
19
Each ofthe test mixtures was composed of 10 mg ofthe corresponding solute
dissolved in 100 mL ofmethanol. All weighings were performed using a Mettler
Balance (Mettler Instrument Corporation, Hightstown, NJ). Each test mixture was stored
in a sealed container at room temperature when not in use.
B. Methods
The synthesis ofthe amino acid-derived t-butylcalix(4)arene was based on a well 
characterized addition ofalanine to calix(4)arene (13). This synthetic method required
significant modification for the addition ofphenylalanine, however. This addition
involved attachment ofphenylalanine residues through the hydroxyl groups at the bottom
ofthe calixarene cup-like structure.
The phenylalanine-derived calix(4)arene was subsequently absorbed into a
dimethylpolysiloxane (nonpolar) stationary phase via a slightly modified static
immobilization method (14). This method involved filling a capillary column with a
dilute solution ofthe phenylalanine derived calix(4)arene, closing one end ofthe column
with a capped reducing union, applying pressure to help drive the calixarene into the
polymer matrix, and subsequently evaporating the solvent under reduced pressure. This
method left behind a coating ofthe chiral calix(4)arene on the inside wall ofthe capillary
column.
1. Synthesis ofthe Chiral Calixarene
The amino acid-derived calix(4)arene was prepared according to a reported
procedure (13), with the following modifications (Figure 3). p-Tertbutylcalix(4)arene
tBu
tBu
tBu
~
NaH
IDMF
~
Me4NOH
~
~
BrCH2COOEtTHF
(1
)
I
CH2COOEt
(2)
(3)
tBu
tButBu
HATUJOIPEAIDMF
~
TFA/H20
~
?
..
*
OCMH2NCHCOOC(CH3h
4
I
o
4
o
4
CH
2
II
?
**
CH2CONCHCOOC(CH3hCH2CONCHCOOH
II
(3)
CH
2
CH
2
66
(4)(5)
Figure
3.Synthesis
of
aminoacid-derivedcalix(4)arene
N
o
21
(0.067g) was suspended in dry DMF (2.1mL) and was treated with NaH (0.025g).
Ethylbromoacetate (0.060mL) was then added and the mixture was stirred at 80?C for 4
hours. The reaction mixture was allowed to cool and was then treated with a second
portion ofNaH (0.015g). Ethylbromoacetate (0.030mL) was then added and the mixture
was stirred again at 80?C for approximately 20 hours to ensure complete alkylation.
The hydrolysis ofthe alkylated p-tertbutylcalix(4)arene was performed by
dissolving the alkylated p-tertbutylcalix(4)arene (O.998g) in THF (50mL). This solution
was added to 10% aqueous tetramethylammonium hydroxide (50mL) and heated at reflux
for 24 hours. After cooling, the reaction mixture was acidified with concentrated HCI
and stirred overnight at room temperature. The resulting bilayered solution was
rotovapped until all ofthe THF was removed, inducing precipitation ofthe hydrolyzed
product. The hydrolyzed product was filtered, washed with water, and oven dried at
100?e.
The addition ofthe amino acid to the hydrolyzed product was performed entirely
under an atmosphere ofargon. All glassware and reagents were dried and additions were
done in such a way as to keep water out ofthe reaction. The hydrolyzed product (l.60g)
was dissolved in dry DMF (80mL) with stirring. HATU (2.75g) and DIPEA (2.80mL)
were added to this solution at room temperature. The resulting solution was cooled to
O?C and L-phenylalanine t-butyl ester HCI (l.87g) was added and allowed to stir at O?C
for the first hour. This solution was allowed to warm to room temperature and stirred
overnight. The product was precipitated out ofsolution by addition ofwater and filtered.
It was subsequently dried under vacuum (the product should not be heated). The addition
product was cleaned by dissolving it into a minimum amount ofa 1: 1 solution ofdistilled
22
THF/ hexanes (reagent grade). This mixture was pushed through a plug ofsilica to
remove impurities. The silica was rinsed several times with the 1: 1 solution to ensure
optimum retainment ofthe product. All ofthese washings were collected in the same
receiving flask and were rotovapped until the solvent was removed, inducing
precipitation ofthe addition product. The addition product was allowed to dry by
vacuum filtration.
The hydrolysis ofthe addition product was performed by dissolving the addition
product (0.100g) in a solution ofTFA containing water (5%) at oac. The solution was
allowed to warm to room temperature where it was stirred for 1 hour. The status ofthe
reaction was checked by TLC (20% THF/hexanes) to ensure completeness ofthe
reaction. Water was then added to the reaction mixture to precipitate the product. The
hydrolyzed amino acid-derived calix(4)arene was filtered and washed with water.
2. Absorption into the Stationary Phase
The absorption ofthe calixarene derivatives was performed according to literature
methods (14) with the following modifications. In a pre-cleaned screw cap with septum
vial, approximately 1 % (w/w) ofthe calixarene derivative was dissolved in methylene
chloride. The mixture was shaken to ensure complete dissolution. The septum vial was
pierced with a 22-gauge needle and the column was fed through the needle into the
solution. The needle was then removed, leaving the column end in solution with an
airtight seal around the column through the septum. The body ofthe column was hung
on a metal rod directly above the vial containing solution. A 20 mL plastic syringe fitted
with a 22-gauge needle was used to supply the column with a small, but constant amount
23
ofpressure. The syringe was prepared by drilling a hole about halfway down the syringe
completely through the barrel and the plunger. To apply pressure onto the solution
(pushing it into the column), the plunger was pulled out as far as possible, and then
inserted through the septum into the vial. Pressure was applied by pushing in the plunger
which was then held in place by placing a small rod through the drilled hole to achieve
constant pressure (Figure 4). After several drops ofthe calixarene solution exited the
outlet end ofthe column, the outlet end ofthe column was sealed by closing one end ofa
1/16" - 1/32" Valco reducing union and attaching the open end to the column with a
1/32" to O.4mm Valco reducing ferrule.
Figure 4. Diagram ofcolumn filling apparatus
3. Evacuation ofthe Solvent
The inlet end ofthe column was removed from the column filling apparatus and
attached to a tank containing ultra high purity N2. Pressure was introduced into the
24
column and gradually increased to 70 psi, where it was held for 20 minutes. This
procedure pushed the calixarene derivative into the silicone polymer stationary phase
attached to the walls ofthe column. The pressure was gradually decreased and the inlet
end ofthe column was placed on a vacuum line and the column was placed in a 30?C
water bath for 40 hours.
4. Conditioning the Column for Use
The inlet end ofthe column was then sealed and the column was preconditioned
by heating at 160?C for 1 hour followed by another hour at 180?C heating. After
preconditioning, the column was mounted on the GC where it was conditioned for 3
hours at 120?C with a carrier gas (He) flow rate of I.OmL/min. The column was cooled
and removed from the GC and stored at room temperature with septa covering both ends.
5. Descriptions ofPrepared Stationary Phases
Columns containing tetra(L-phenylalanyl)-4-tetra(tert-Butyl)-I-
tetra(carboxymethyl)calix(4)arene tetra(tert-Butyl)ester were prepared along with
columns containing tetra(L-phenylalanyl)-4-tetra(tert-Butyl)-I-tetra(carboxymethyl)
calix(4)arene. This was done to investigate the differences in the properties ofthe
stationary phases due to drastically different polarities and to also help in the overall
characterization ofthe stationary phases. The underivatized AT-1 stationary phase was
used as a reference for comparison ofthe derived stationary phases.
25
6. GC Parameters
The GC unit consisted ofa Varian (Walnut Creek, CA) 3400 CX Gas
Chromatograph with Star Chromatography (Version 4.01) software equipped with a
flame ionization detector (FID). The capillary column used as a reference was an Alltech
Heliflex AT-l (Deerfield, IL) 10m x 0.25mm ID x 0.25~m. The derivatized stationary
phases were absorptions onto this AT-1 column. Table I lists the chromatographic
operation conditions used for characterization ofthe stationary phases.
Table 1. Typical Chromatographic Conditions
Column: Alltech AT-1 (catalog number 13753),
10m x O.25mm 10 x O.25/lm
Column temperature: 90?C
Sample concentration: - 300ng/ml in MeOH
Injection volume: 1 IlL
Detector: Flame Ionization Detector
Flow rate: 1.0 mLlmin with He as carrier gas
Stationary phase A: 1 wt. % unhydrolyzed a.a.-derived product in CH2CI2
Stationary phase B: 1 wt. % hydrolyzed a.a.-derived product in CH2CI2
7. GC Experiments
Chromatographic separations were performed on a series ofdifferent solute
mixtures. Each mixture ofsolutes was specifically chosen to determine the physical
characteristics ofthe column. These characteristics included how the stationary phases
26
behaved with regard to van der Waals interactions (polarity effects), differences in peak
shape and separation efficiency (H-bonding effects), indications ofintermolecular
interactions (7t-7t interactions), and to determine ifhost/guest complexation was occurring
between the solute and the stationary phase. A reference homologous series ofalkyl
benzenes (RHS) comprised oftoluene, ethyl benzene, propyl benzene, and butyl benzene
was separated in order to characterize the 7t-7t interactions and to determine ifhost-guest
complexation occurred between the calixarene and phenyl containing compounds. A
homologous series ofalcohols (AHS) comprised of I-propanol, I-butanol, I-pentanol,
and I-hexanol was used to look at the effects ofH-bonding between components and the
stationary phase and to see ofhost-guest complexation would occur through H-bond
interactions at the derivatized bottom ofthe cup-like structure. A series ofphenolic
compounds, which contained phenol, resorcinol, and tert-butylphenol, was used to
determine the effects ofH-bonding and to indicate any 7t-7t interactions and host-guest
complexation. Two series ofisomeric compounds containing 2-methyl-2-butanol, 3 
methyl-2-butanol, and 2-pentanol along with tert-butanol, sec-butanol, and n-butanol
were also used to determine the effects ofH-bonding. Finally, a mixture containing a 
and p-napthol and (-)-menthol was used to characterize H-bonding effects, 7t-7t
interactions, and the effects ofpolarity upon elution ofcomponents between the different
stationary phases, and host-guest interactions.
27
CHAPTER V
Results and Discussion
A. Characterization ofthe Chiral Phenylalanine-Derived Calix(4)arene
The addition step for the unhydrolyzed product characterized here was described
by Pena et al (21) with modification. The modification involved the addition ofL 
phenylalanine t-butyl ester HCl instead ofalanine to the bottom ofthe calix(4)arene
structure. Also, the addition product ofthis reaction was purified by dissolving it into
warm methanol. Precipitation ofthe product was induced by the addition ofwater. TLC
plates were run in THF and showed only a single product. The I H NMR spectrum
(Figure 5) in d-chloroform showed only minor impurities. A singlet at 1.1 ppm and a
singlet at 1.3 ppm were obtained for protons at the 1 and 9 positions, respectively. Also,
singlets indicated protons at position 3 (1.4 ppm), protons at position 2 (6.7 ppm), and
protons located at position 8 (7.2 ppm). A quartet located at 3.7 ppm was obtained for
protons located at position 6. Multiplets were obtained for protons located at position 7
(3.1 ppm), protons at position 4 (4.5 ppm), and protons at position 5 (7.8 ppm).
The successful synthesis ofthis product was supported by mass spectral data
(Figure 6) obtained from a Bruker Esquire LCMS. Atmospheric pressure chemical
ionization (APCI) ionized the compound and the ions were mass-analyzed with an ion
trap. A scan range ofm/z 200 - 2200 was used with an accumulation time of426 J.!S and
the polarity set to positive. The [M+1] peak that was obtained was at m/z 1695, which
corresponds to that ofthe addition product.
(9)(1)
(1)
o
'40
I
(6)
II
+
(4)H
2
C-C-NH-CH-C-O
9)
II
(5)
I
o
6
m
(8)
(3)(7)(6)
(4)
(2)
8)
(5)
I
I
-,--
II
12
1086420
ppm
Figure5.
IH
NMR
ofunhydrolyzedaminoacid-derivedcalix(4)arene
tv
00
Intens.
x10
7
1695.1
4
3
210
l
25050075010001250150017502000
m/z
Figure
6.Massspectrum
of
unhydrolyzedproduct
tv
\0
30
The hydrolysis ofthe addition step (hydrolyzed product) characterized here was also
described by Pena et al (21) with modification. The hydrolysis was performed with TFA
and 5% water, without the DCM called for in the literature. TLC plates were run with
20% THF in hexanes and indicated only a single product. The IH NMR spectrum (Figure
7) ofthe product in d-chloroform showed minor impurities. Protons located at positions
1 and 3 resulted in singlets found at 1.0 ppm and 2.5 ppm, respectively. A multiplet was
obtained at 3.0 ppm for protons located at position 7, at 4.4 ppm for protons located at
position 4, at 4.6 ppm for protons located at position 6, at 6.7 ppm for protons located at
position 2, at 7.2 ppm for protons located at position 8, and at 8.5 ppm for protons located
at position 5. A broad peak very characteristic ofa hydroxyl group was obtained for the
protons located at position 9 were found at 5.0 ppm.
The synthesis ofthis product was supported by APCI mass spectral data (Figure
8). A scan range from m/z 200 - 2200 was used with an accumulation time of 1994 /lS
and the polarity set to positive. The [M+1] peak that was obtained was at m/z 1471,
which corresponds to that ofthe final hydrolyzed addition product.
B. Absorbed Stationary Phases
The data presented in this work was obtained for three types ofstationary phases.
The columns used contained an AT-1 stationary phase (AT-1), a column with tetra(L 
phenylalanyl)-4-tetra(tert-Butyl)-tetra(carboxymethyl)calix(4)arene tetra(tert-Butyl)ester
absorbed into an AT-1 siloxane matrix (unhydrolyzed), and a column with
(3)
(l)
(1)
o
'4
0
I
(6)
II
(4)
H
2
C-C-
NH-CH-C-OH(9)
II
(5)
I
o
6(7)
(8)
(8)
(2)
(5)
J
-------r--
-----.-------r-I
i
12
10
8
(7)
I
ppm
Figure
7.
lH
NMR
of
hydrolyzedaminoacid-derivedcalix(4)arene
w
Intens.
x10
6
1470.7
1.2
1.0
0.8
0.60.4
0.2
10.91276.7
0.0
It
.
U
,d
,I.
II
J.
1.
.~...
L
I,ll
L
lid
lilt
J
II
II.
1.?\
.1
25050075010001250150017502000
m/z
Figure
8.
Massspectrum
of
hydrolyzedproduct
VJ
tv
33
tetra(L-phenylalanyI)-4-tetra(tert-Buty1)-tetra(carboxymethyI)calix(4)arene absorbed into
an AT-1 siloxane matrix (hydrolyzed).
C. Homologous Series ofAlkyl Benzenes
Calixarenes participate in host-guest interactions with molecules that are aromatic
or contain aromatic functionality(s). A data summary for the RHS is found in Table II.
Comparison ofthe retention data before and after derivatization with both the hydrolyzed
and the unhydrolyzed form ofthe amino acid-derived calix(4)arene characterized any
formation ofhost-guest interactions between the stationary phase and the RHS-.
Interactions based solely on polarity resulted in a linear relationship and any deviation
from linearity indicated the presence ofsome 7t-7t interaction (25).
Figure 9 shows a comparison ofthe retention behavior observed with both the
hydrolyzed and the unhydrolyzed stationary phases along with the linear dependency of
the retention data for RHS on the underivatized AT-1 stationary phase. There was some
evidence ofinteraction between solute and the unhydrolyzed amino acid-derived
stationary phase based on its correlation value ofR=O.9943, where the correlation for the
AT-l and hydrolyzed amino acid-derived stationary phase were R=O.9998 and R=O.9997,
respectively.
Along with host-guest interaction information, Figure 9 also shows the relative
polarities ofall stationary phases before and after derivatization. The hydrolyzed product
resulted in a stationary phase more polar than the reference AT-1 phase, therefore the
retention times associated with it were smaller compared to the AT-1 reference. The
34
unhydrolyzed stationary phase resulted in a more non-polar stationary phase, therefore
the retention times associated with it were larger than the AT-1 reference.
The RHS was also used as a reference to determine the durability ofthe column.
It determined ifthe behavior ofthe stationary phases had been altered by either a
chemical reaction or degradation from heat. Duplicate trials were performed as the first
and last assays on all columns and identical chromatograms were obtained indicating that
little or no change had occurred in the chemical characteristics ofthe stationary phases.
D. Homologous Series ofAlcohols
The AHS was separated to help determine ifH-bonding was occurring, which
would result in extended retention from adsorption ofcomponents with hydroxyl
functional groups. Figure 10 shows the van't Hoffplot ofIn k' vs. the number ofcarbons
in the AHS where deviation in linearity would indicate the presence ofsome interaction
resulting from H-bonding. The correlation values for the AT-1 (R=0.99996), the
hydrolyzed (R=0.99996), and the unhydrolyzed (R=0.99998) stationary phases indicated
no significant additional interactions (i.e., due to host-guest formation) between the
aliphatic alcohols and either the hydrolyzed or unhydrolyzed stationary phases. Also, the
contrast in the differences in retention due to differences in polarity between stationary
phases was not as apparent here as with the RHS. Further investigation into H-bonding
effects was performed by calculation ofthe capacity factors (k'), where an increase
would indicate some interaction. Table II summarizes the chromatographic data obtained
from elution ofthe AHS. k' between the AT-l vs. the unhydrolyzed stationary
4.54
C)
3.5
00
E
0
3
:::c
.E
(/)
t:
0
.0
...
CO
U
-
0
...
Q)
.0
E
::J
Z
0.5
-+-AT-1
-Hydrolyzed
-+-
Unhydrolyzed
-0.6-0.4-0.2
o
log
k'
0.2
0.4
0.6
Figure
9.Effects
of
host/guestcomplexationuponcorrelation
of
referencehomologousseriesat90?C
w
VI
6.5
3
--
-
5.5
C)
00
E
0
."
~
5
:::I:
c
VI
C
0
/
--+-AT-1
4.5
.0
"-
CO
--
Hydrolyzed
U
-
...........
Unhydrolyzed
0
4
"-
Q)
.0
E
::::I
Z
-/
./
/
3.5
-1.2
-1
-0.8
-0.6
-0.4
-0.2
o
log
k'
Figure
10.Effects
of
host!guestcomplexationuponcorrelation
of
alcoholhomologousseriesat90?C
IN
0\
QxrpolnI
#dcarbalS
tr*(AT-1)
t.-*(~.)
tr*(unpoI.}
k'
(AT-1)
k'(~.)
k'(unpoI.}
tdl..Ere
7
0.1974
0.1811
0.34ffi
o.E12
?
1.F/o
0.3811
?
1.130/0
0.49DJ?
o.2EIP/o
~t::x;lIzBe
8
0.3704
0.34a5
0.fB70
0.
72f5Zl
?
o.fffilfo
0.71654?0.418%
0.ffi2ffi
?
0.182%
p-q::?
I:a
IZB
e
9
0.EB31
0.6291
1.0400
1.3431
?
0.28Y1o
1.32383
?
0.:r04%
1.49429
?
0.128%
~l:alZBe
10
1.32931.2247
1.9752
261463?
0.1CXWo
25T7Z3
?
0.127%
282171
?o.OO7%
1-pq:xrd3
0.:D14
0.2331
0.4438
0.ff975?1.11%
0.48431
?
HE%
0.
71
em
?
0.a:a3%
1-b.ta"d4
0.ffi32
0.ffi37
0.9331
1.32146
?
1.03%
1.13579
?
0.857'%
1.49200
?
0.638%
1-prtcrd5
1.50C6
1.
3m
22210
3.ZJTl7
?
o.Effi%
2ffi7CE
?
0.745%
3.55.E?
0.f64%
1-rea-d
6
4.04a5
3.0078
5.5751
8.17419
?
0.781%
7.4aE2?
o.77fflo
8.9Z>16
?
0.492'%
2-rrett"?-2-b.ta"d
5
0.4OCl8
0.4328
0.8222
1.01411
?0.4ffi%
o.~
?
0.ffi30/0
1.32849
?
0.5EOlfo
~-2-b.ta"d
5
0.71030.6d57
1.1:;E8
1.452ffi
?
0.524%
1.31246
?
0.942%
1.~
?
o.!XF/o
2-p:.rtad5
0.83750.7402
1.3)44
1.
71313
?
0.500%
1.50016
?
mm%
210761
?
0.533%
tr*
is
correctedcomponentretentiontime
(mi,n)
Table
2.
ComponentRetentionData
w
-.l
38
phases shows an increase which suggests an increase ofinteraction due to polarity
effects. The slight curvature ofthis plot may suggest a hint ofhost-guest interaction, but
it is much smaller than that seen with the RHS. These differences in retention are due
mostly to polarity differences.
E. Phenolic Mixture
This phenolic mixture was used to determine the effects ofH-bonding and to
indicate any host-guest formation and/or 7t-7t interactions. Figure 11 shows the elution of
this mixture on all stationary phases at 80?C. All components eluted symmetrically from
the AT-l within approximately 12 minutes. On the hydrolyzed and unhydrolyzed
stationary phases, phenol tailed significantly strongly indicating a host-guest interaction.
The resorcinol never eluted from either ofthe derived stationary phases, also indicating a
very strong interaction. The tert-butylphenol did not tail, the only differences upon
elution being due to the polarity differences that resulted from the derivatization of
stationary phases, not from host-guest interaction. This chromatogram gave an indication
ofthe relative component polarity necessary for interaction and the size ofthe inner
cavity created by derivatization ofthe amino acid-derived calix(4)arene.
F. Isomeric Mixtures
The isomeric mixtures were separated to help determine the effects ofvarious
isomeric positions ofthe hydroxyl group upon that component's ability to H-bond with
the stationary phase. Figure 12 depicts a chromatogram oftert-, sec-, and n-butanol,
which showed no selectivity toward any ofthe isomers. The differences in retention
(3)
(1
)
(3)
'AT-1HydrolyzedUnhydrolyzed
3
579
11
time(min)
13
15
17
19
Figure11.
Retention
of
phenol(1),resorcinol(2),
&
tert-butylphenol(3)at80?C
w
\0
.,..
AT-1
r-t=/\
Hydrolyzed
,--~~~-,~~~~-,-~~~~---,--~~--===r--~~~-=r====--,~~~---===r==="""""'iIUnhydrolyzed
0.7
0.80.9
1.1
time(min)
1.2
1.3
1.4
1.5
Figure12.Retentionoftert-butanol,sec-butanol,andn-butanol(respectively)at
3fc
+> 
o
41
were primarily due to differences in the polarities ofthe stationary phases. The tailing of
the components as eluted from the derived stationary phases could possibly indicate a
small amount ofhost/guest complexation. Table III summarizes the asymmetry factors
for this isomeric mixture and shows an increase in A for each component when compared
to that for the AT-1 Reference.
Compound tr k' Asymetry Factor
a-napthol (AT-1 ) 22.6357 44.3258 ? 0.014% 1.10
a-napthol (Hydrolyzed) 20.4467 39.4725 ? 0.020% 3.67
a-napthol (Unhydrolyzed) 29.3901 40.5230 ? 0.147% 1.83
~-napthol (AT-1) 23.6944 46.9256 ? 0.054% 1.30
~-napthol (Hydrolyzed) 21.6217 41.7983 ? 0.308% 17.0
~-napthol (Unhydrolyzed) 30.7508 42.4456 ? 0.047% 5.18
n-butanol (AT-1 ) 0.6565 1.33598 ? 0.826% 0.75
n-butanol (Hydrolyzed) 0.5625 1.15101 ? 0.266% 2.42
n-butanol (Unhydrolyzed) 0.9755 1.55681 ? 0.644% 1.14
sec-butanol (AT-1 ) 0.5278 1.07407 ? 0.836% 0.68
sec-butanol (Hydrolyzed) 0.4487 0.91815 ? 0.208% 0.71
sec-butanol (Unhydrolyzed) 0.8027 1.28104 ? 0.669% 0.63
tert-butanol (AT-1 ) 0.3957 0.80525 ? 0.933% 0.43
tert-butanol (Hydrolyzed) 0.3268 0.66871 ? 0.232% 1.07
tert-butanol (Unhydrolyzed) 0.6160 0.98308 ? 0.742% 0.64
Table 3. Asymmetry Factors
Figure 13 shows the chromatogram of(-)-menthol, which is a large alcohol. This
component eluted as symmetrical peaks indicating little interaction due to H-bonding.
2
\
2.2
2.4
2.62.833.2
3.4
3.63.844.2
4.4
4.64.8
time
(min)
\
-AT-1
-
Hydrolyzed
-
Unhydrolyzed
55.2
5.4
5.65.86
Figure13.Retention
of
(-)-menthol
at
90?C
~
~
N
N
~
I
~
I
\--...J
--.J
AT-1Hydrolyzed
0.7
0.9
1.1
1.3
time(min)
1.5
1.7
1.9
Unhydrolyzed
2.1
Figure14.
Retention
of
2-methyl-2-butanol,3-methyl-2-butanol,and2-pentanol(respectively)at35?C
~
w
44
Eluting another isomeric mixture containing 2-methyl-2-butanol, 3-methyl-2-butanol, and
2-pentanol allowed further investigations into any H-bonding effects. Figure 9 shows the
chromatogram ofthis series and depicts no selectivity ofisomers with the only
differences upon elution being the differences in polarity. The capacity factors (k') for
this series were calculated, where an increase indicated an interaction. Table II
summarizes the component retention data obtained from elution ofthis series, which
shows an increase in k' between the AT-1 vs. the unhydrolyzed stationary phase. This
suggests an increase in interaction ofsolutes and the unhydrolyzed stationary phase.
G. Mixture ofa- and I3-Napthol
A mixture ofa-, l3-napthol was chosen to determine ifthe components were
participating in any type of1t-1t interactions, ifany H-bonding was occurring, and the
effects ofsize and polarity ofthe component upon host-guest formation. Figure 15
shows the chromatogram ofthe napthols, in which the relative polarities ofthe stationary
phases can be seen. This data demonstrates the ability ofthe stationary phases to
selectively interact with a particular component based upon its size and relative polarity.
Table III gives the asymmetry factors that were determined for the napthols. The
increasing values for the asymmetry factors for these components strongly suggest
interactions between some solutes and both amino acid-derived calix(4)arene
stationary phases are occurring. This data also indicated that this newly derived
stationary phase is capable ofselectively participating in the host-guest interactions with
a solute based on relative size and polarity, as the elution ofnapthol resulted in
45
significant tailing ofcomponents, while the elution of(-)-menthol resulted in symmetrical
peaks.
(1
)
(1
)
(1
)
(2)
\,
-+
\
,.}.
AT-1
...
'*'
F"
I
I
c
u
0"
'
~
riM
n
4('"
t
?'~
1,1
,.
???
" ?
-.
"lee
of
I
ill"
_.'
..
PIN
P ,
WM...
I
.:t
~
~
~
~
~
I
Hydrolyzed
~
.........
,.'"
'~~
...
','
T.
I'M
....
tUI,dt
??
r
18
20
22
24 26
28
time (min)
30
32
34 36
38
Figure
15. Retention
of
a-napthol
(1)
and l3-napthol (2) at 90?C
+:0  0"1
47
CHAPTER VI
Conclusions
This research has demonstrated the successful synthesis of L-phenylalanine
derived 4-t-butylcalix(4)arene and its ability to participate in host-guest interactions with
particular solutes in gas chromatography. The derivatized chiral calix(4)arene was
absorbed onto a non-polar stationary phase, and its behavior with respect to polarity and
component size dependence is described in this work.
The synthesis ofthe L-phenylalanine derived 4-t-butylcalix(4)arene was
successful, as supported by mass spectral and I H NMR data along with the retention
behavior ofthe solutes on the derivatized stationary phases. The increase in the capacity
factor (k') and the increased values for the asymmetry factors strongly suggest host/guest
interactions between some solutes and the immobilized calixarene. The differences in the
relative polarities ofthe stationary phases are reflected in the retention times ofthe
solutes. The hydrolyzed derivative increases the polarity and the unhydrolyzed
derivative decreases the polarity with respect to the underived AT-1 stationary phase.
The asymmetry factors for the chromatographic peaks ofthe (-)-menthol vs. that for
napthol show that this chiral calix(4)arene can specify the host/guest interactions in
which it participates. The host/guest interactions ofthe L-phenylalanine derived 4+
butylcalix(4)arene may lead to the additional chromatographic dependence from which
enantiomeric separations can be performed.
(1
)
(1
)
(1
)
38
34
36
----"
t
"$
S ?
n
7
Unhydrolyzed
?
un
....
'an
32
(2)
_.
..
,
......
'
...
,.
~:
::::
':
':::::t:
:!ts;;:::
::::':::
:::::
::~"O.Z.d
??
I
..
t'"
(s
II
U
OW,'
g'
It
,....J
18
20
22
24
26
28
30
time (min)
Figure 15.
Retention
of
a-napthol (1) and
~-napthol
(2) at 90?C
~
47
CHAPTER VI
Conclusions
This research has demonstrated the successful synthesis of L-phenylalanine
derived 4-t-butylcalix(4)arene and its ability to participate in host-guest interactions with
particular solutes in gas chromatography. The derivatized chiral calix(4)arene was
absorbed onto a non-polar stationary phase, and its behavior with respect to polarity and
component size dependence is described in this work.
The synthesis ofthe L-phenylalanine derived 4-t-butylcalix(4)arene was
successful, as supported by mass spectral and 1H NMR data along with the retention
behavior ofthe solutes on the derivatized stationary phases. The increase in the capacity
factor (k') and the increased values for the asymmetry factors strongly suggest host/guest
interactions between some solutes and the immobilized calixarene. The differences in the
relative polarities ofthe stationary phases are reflected in the retention times ofthe
solutes. The hydrolyzed derivative increases the polarity and the unhydrolyzed
derivative decreases the polarity with respect to the underived AT-1 stationary phase.
The asymmetry factors for the chromatographic peaks ofthe (-)-menthol vs. that for
napthol show that this chiral calix(4)arene can specify the host/guest interactions in
which it participates. The host/guest interactions ofthe L-phenylalanine derived 4-t 
butylcalix(4)arene may lead to the additional chromatographic dependence from which
enantiomeric separations can be performed.
48
CHAPTER VII
Future Work
A focus offuture work should involve investigating the upper temperature limits
ofthe derived stationary phases. All effects occurring from these columns seemed to be
enhanced with temperature, so simply being performed at higher temperatures could
potentially optimize some effects.
A study involving the refinement ofthe synthetic procedure that was roughly
mapped out in this research should be performed. Purification ofthis novel crural
calixarene step-by-step gives the researcher insight into the relative polarity ofthis
compound, along with a higher percent recovery offinal product. A better understanding
ofthe relative polarity ofthis compound and the stationary phase that results from it will
draw a clearer picture ofthe types ofcomponents this stationary phase would prefer
interaction with.
Exploring the behavior ofother test mixtures, (i.e., aceto- through benzonitrile) to
give more insight into the size and polarity ofthe cavity will help determine the types of
compounds these columns could potentially show enantiospecificity towards.
Calculation and comparison ofthe binding coefficients ofthe RHS between columns
could provide interesting results.
Future investigations should also include making a chemical attachment ofthe
chiral compound to the inner wall ofthe column and determining the temperature limits
and effects ofthis adsorption. Also, investigations should be made into the application of
the synthetic methods described in this work to the addition ofother chiral amino acids to
49
calix(4)arene to determine the effects ofother amino acid-derived calix(4)arenes to host 
guest complexation.
Additional investigations should be performed for determining the effects ofthe
thickness ofthe coating ofchiral calixarene deposited on the column after the absorption
process. Simply dissolving more ofthe amino acid-derived calixarene in DCM before
introducing the solution into the column could easily increase the concentration ofthe
coating. This increase in coating thickness should result in an increase in the amount of
host-guest interaction between solute and stationary phase, which would be reflected in
increased values for the asymmetry factors for the injected components. Optimizing
these interactions will hopefully lead to a better understanding ofthe enantiomeric
separations in which this type ofstationary phase can be applied. Also, an increase in the
asymmetry factors ofthe components upon increased concentration ofthe coating in the
column would provide further evidence ofhost-guest complexation.
50
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51